Malignant mesothelioma (MM) is an aggressive, treatment-resistant cancer linked to asbestos exposure. The genetic basis for MM has historically focused on somatic mutations of CDKN2A and NF2 as key alterations influencing initiation and progression. Very recently, the BAP1 ubiquitin carboxy-terminal hydrolase, first isolated as a Ch. 3p21 tumor suppressor in 1998 (Rauscher Lab), has been strongly implicated as a major player in MM based on genetic analyses. Germline BAP1 mutations were found in two families with a high incidence of MM and other cancers, and somatic BAP1 alterations occurred in MMs, consistent with biallelic inactivation of a tumor suppressor (Testa Lab). Moreover, somatic BAP1 mutations are common in sporadic MMs and in both sporadic and familial uveal melanoma (UM). The genetic and biochemical mechanisms by which BAP1 mutations predispose to MM and UM and how BAP1 interacts genetically with CDKN2A and NF2 to influence MM pathology, prognosis, and therapeutic response are largely unknown. However, the fact that BAP1 encodes a nuclear-localized de-ubiquitinase that binds to ASXL1/2, an obligate polycomb family partner protein that targets histones, strongly suggests a role in epigenetic regulation of gene transcription. Moreover, since binding of BAP1 to ASXL1/2 is required for enzyme activity and tumor suppression, an alternative way to inactivate BAP1 tumor suppressor activity may be to mutate or silence ASXL1/2 genes. To define both the biochemical mechanisms and the genetic interactions among BAP1, ASXL1/2, CDKN2A and NF2 in MM, the Rauscher and Testa Labs have joined forces to pursue the following Specific Aims: 1) Use a direct in vivo genetic approach to determine if heterozygous Bap1-mutant (+/mut) mice are predisposed to the development of various spontaneous tumors, including MM, and accelerate asbestos-induced MM. 2) Use conditional knockout mice to determine if somatic loss of Bap1 alone in the mesothelium is capable of driving mesothelial hyperplasia and/or frank MM, and if somatic Bap1 inactivation combined with loss of Nf2 and Cdkn2a results in more rapid development and/or a more aggressive disease phenotype, with potential prognostic and novel therapeutic implications. 3) Define the spectrum of BAP1 and ASXL1/2 mutations in human MM specimens/cell lines and determine how these mutations affect binding and function of BAP1 in complex with ASXL1/2 and other PR-DUB complex proteins, and define the transcriptome and cellular phenotype which are altered as a consequence of changes (via knockdown and reconstitution) in BAP1 and other PR-DUB components in MM cells. 4) Determine the composition of the native PR-DUB macromolecular protein complexes that contain wild- type BAP1 and ASXL1/2 in mesothelial cells and in BAP1- and/or ASXL1/2-deficient MM cells to compare normal- and tumor-specific subunit structure and the role of these complexes in epigenetic dysregulation. The proposed multipronged studies by two PIs with complementary expertise represent a comprehensive approach to yield novel insights into MM pathogenesis while providing new targets for therapy and prevention.